To investigate the potential distribution of radiolabelled botulinum neurotoxin type A (BoNT/A) in the CNS after bladder injection in normal rats, by using the gamma-emitting radionuclide technetium-99 m ((99m) Tc).,BoNT/A was radiolabelled by pretreatment with 2-iminothiolane and incubation with (99m) Tc-gluconate. The labelled toxin (99m) Tc-BoNT/A was purified using size exclusion HPLC. Twenty-four female Wistar rats were evenly injected in the bladder wall with either (99m) Tc-/ (n = 12) or free (99m) Tc (n = 12). Four rats from each group were killed at 1, 3 and 6 h after injection, respectively. The bladder, L6-S1 spinal cord segment and L6-S1 dorsal root ganglia (DRG) were harvested and their radioactivity counted in a gamma scintillation detector. Results were calculated as % injected dose (I.D.) per gram of tissue. The paired t-test was used for comparison of means of (99m) Tc-/ radioactivity vs free (99m) Tc in the tissues of interest.,Radiolabelled BoNT/A had a high radiochemical stability of 70% after 24 h. Gradual accumulation of (99m) Tc-/ was observed in the DRG up to 6 h after injection (P = 0.04 and P = 0.029 compared with 1 h and 3 h, respectively), while no accumulation was detected for free (99m) Tc. Consequently, (99m) Tc-/ radioactivity in the DRG was higher than free (99m) Tc radioactivity (3.18 0.67% I.D./g vs 0.19 0.10% I.D./g [P = 0.002] 6 h after injection). Values for (99m) Tc-/ radioactivity in the spinal cord were higher than those for free (99m) Tc, but not significantly. The bladder retained higher dosages of (99m) Tc-/ than free (99m) Tc at all time points.,Significant accumulation of the radiolabelled toxin in the lumbosacral DRG, together with a less significant uptake in the respective spinal cord segment as opposed to free radioactivity provide first evidence of the retrograde transport of BoNT/A to the CNS after bladder injection in rats.